Myeloproliferative neoplasms (MPNs), formerly classified as chronic myeloproliferative diseases, are clonal stem cell disorders characterized by proliferation of 1 or more of the myeloid lineages (granulocytic, erythroid, mast cell, or megakaryocytic). These neoplasms collectively have an incidence of 6 to 10 per 100,000 population annually.¹ This Clinical Focus describes the various MPNs and the use of laboratory testing for diagnosis and management.

The classic, more common MPNs include chronic myelogenous leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Chronic eosinophilic leukemia, not otherwise specified (CEL, NOS), systemic mastocytosis (SM), chronic neutrophilic leukemia (CNL), and unclassifiable MPN are rare. MPNs typically occur in adults 50 to 70 years old and are uncommon in individuals <20 years old. Frequently, the onset is insidious and the clinical course indolent. Patient complaints may include fatigue and lethargy, weight loss, abdominal discomfort, easy bruising, night sweats, and swollen, painful joints. Physical examination may reveal pallor, enlargement of the spleen or liver, and petechiae.

Distinguishing between the MPNs is often difficult because of the overlap of clinical and laboratory findings. For example, most MPNs result in increased numbers of granulocytes, RBCs, and/or platelets. Each MPN begins with effective hematopoiesis resulting in circulating mature blood cells, but may result in marrow failure due to myelofibrosis, ineffective hematopoiesis, or progression to acute myeloid leukemia (AML). Table 1 details hematologic characteristics of the various MPNs, including those considered diagnostic by the World Health Organization (WHO).

Chronic Myelogenous Leukemia

Patients may present with splenomegaly, but more commonly CML is detected with increased WBC count and percent neutrophils in asymptomatic patients. The hallmark of CML is the presence of the Philadelphia chromosome (Ph) and/or the BCR/ABL1 fusion gene.² CML progresses through 2 or 3 sequential phases: 1) chronic phase in which most patients are diagnosed, 2) accelerated phase, and/or 3) blast phase. Each phase is progressively more resistant to therapy, and thus, early diagnosis and treatment is imperative.

Essential Thrombocythemia

Although some patients present with symptoms of vascular occlusion or hemorrhage, ET is asymptomatic in more than 50% of patients and is identified fortuitously with a routine CBC that reveals an elevated platelet count.³ Thrombocytosis due to secondary causes such as systemic infections, inflammatory conditions, bleeding, or malignancy must be ruled out before clonal ET can be diagnosed. Thrombosis and hemorrhage are the most frequent clinical complications in patients with ET. The use of cytoreductive therapy to reduce elevated platelet counts has been suggested for high-risk patients (ie, age ≥60 years or a history of thrombosis).⁹

Polycythemia Vera

The most serious complications of PV are thrombosis, hemorrhage, and hypertension. Approximately 20% of patients present with venous or arterial thrombosis, myocardial ischemia, or stroke.⁴ Major complaints at diagnosis include headache, dizziness, visual disturbances, and numbness/tingling. Erythromelalgia (vasodilation with burning), pruritus, and gout may also be present. About 70% of patients have plethora or splenomegaly and 40% have hepatomegaly.⁴ Low dose aspirin and phlebotomy targeting reduction in hematocrit are typically used to treat PV.

Primary Myelofibrosis

PMF, previously known as agnogenic myeloid metaplasia, is characterized by anemia, progressive splenomegaly and bone marrow fibrosis, and multi-organ extramedullary hematopoiesis (EMH). Up to 30% of patients with PMF are asymptomatic at diagnosis, and CBC findings or splenomegaly seen during a routine physical examination trigger the diagnostic workup.⁵ The remainder present with signs of EMH, which accounts for many of the peripheral blood findings in fibrotic PMF. Major causes of morbidity and mortality include AML, which develops in 5% to 30% of PMF patients; bone marrow failure due to hemorrhage or infection; thromboembolic events; portal hypertension; and cardiac failure.⁵

Chronic Eosinophilic Leukemia

In 2008, the WHO categorized neoplastic eosinophilia into 2 classes: 1) a MPN called CEL, NOS that is derived from a myeloid stem cell and 2) eosinophilia associated with abnormalities of PDGFRA, PDGFRB, or FGFR1, all of which are derived from a mutated lymphoid-myeloid stem cell. Clinical features are similar between the 2 classes. Sometimes hypereosinophilia is detected incidentally and no symptoms are apparent.⁶ Other patients may experience constitutional symptoms including fever, fatigue, cough, angioedema, muscle pains, pruritus, and diarrhea. Tissue infiltration by eosinophils, especially in the heart, skin, nervous system, and lungs, may lead to more serious symptoms. Organ involvement, especially in the heart, is the most severe complication.

Systemic Mastocytosis

Clinical symptoms of SM can be grouped into 1) constitutional symptoms; 2) skin conditions such as pruritus and urticaria; 3) mast cell mediator-related features including abdominal pain, flushing, headache, and respiratory symptoms; and 4) musculoskeletal complaints including arthralgia, myalgia, and bone pain and fractures.⁷ Symptoms range from mild to life-threatening depending on the degree of organ involvement. SM should be distinguished from cutaneous mastocytosis, a childhood disorder usually confined to the skin that frequently shows spontaneous regression.

Chronic Neutrophilic Leukemia

Splenomegaly, the most constant clinical feature, is caused by neutrophilic infiltration and may be symptomatic.⁸ Most patients have hepatomegaly, and 25% to 30% report a history of bleeding.

• Individuals with hematologic abnormalities, with or without clinical symptoms of MPNs (eg, organomegaly, vascular occlusion or hemorrhage)

• Individuals being monitored following diagnosis of a MPN

Table 2 lists tests used for diagnosis and management of MPNs.

Bone Marrow Histology

WHO diagnostic guidelines include bone marrow findings as criteria for identifying and distinguishing between the MPNs (Table 1). The WHO recommends that bone marrow biopsy and peripheral blood specimens be evaluated together to reach a diagnosis.¹ Quest Diagnostics uses various stains and immunohistochemical markers to evaluate bone marrow. Cellularity, collagen and reticulin fibrosis, and proliferation of granulocytes, megakaryocytes, mast cells, and erythrocytes are routinely evaluated to aid in the diagnosis of a MPN.

MPN progression is associated with increased bone marrow fibrosis and transformation to AML.
Thus, once a MPN has been diagnosed, disease progression may be monitored with periodic evaluation of bone marrow cellularity, degree of fibrosis, and cytogenetic changes. A more aggressive clinical course is predicted by ≥20 myeloblasts; morphologic evidence of dysplasia; and/or increased reticulin fibrosis, followed by collagen fibrosis, which may result in marrow failure.

Genetic Abnormalities

Because of the clonal nature of the MPNs, detecting chromosomal abnormalities and somatic mutations is important for diagnosis, treatment selection, and monitoring. In the case of CML, a specific chromosomal abnormality (ie, Ph+) is considered diagnostic (Table 1). No other single chromosomal abnormality or molecular marker is solely diagnostic for the other MPNs. However, recurring abnormalities that are not disease-specific have been associated with the non-CML disorders (Table 3). Detection of these abnormalities meets the WHO requirement for establishing clonality and thus supports the diagnosis of a MPN.¹ Additionally, identification of a chromosomal abnormality rules out a non-malignant reactive disorder.

Comparing baseline bone marrow karyotyping with subsequent karyotyping is used to identify clonal evolution, which is the appearance of a genetic abnormality not present previously. Clonal evolution is associated with a poor prognosis and, in CML, is indicative of passing from the chronic to the accelerated or blast phase (Table 3).

Identification of MPN-associated somatic mutations (Table 4) also meets the WHO diagnostic criterion for establishing clonality and rules out reactive causes of erythrocytosis, myelofibrosis, or thrombocytosis. These mutations are associated with the constitutive (unregulated) activation of a protein-tyrosine kinase (PTK) that is implicated in the pathogenesis of each of the MPNs. Thus, drugs targeting PTK activity, such as imatinib, have been successfully used to treat patients with a MPN.¹⁵

JAK2 Mutations

JAK2 V617F is the first acquired gain-of-function mutation found to be associated with PV, PMF, and ET; its detection supports the diagnosis of these MPNs but does not distinguish between them. A negative result does not rule out the diagnosis, however, because the mutation is not always present (Table 4). In JAK2 V617F-negative patients suspected of PV, JAK2 exon 12 mutations should be sought, especially when erythrocytosis is present.¹² Several clinical studies have indicated that the JAK2 V617F mutation is not present in patients with CML, secondary erythrocytosis, SM, or normal control subjects,^16,17 but is present in up to 2% of de novo AML cases.¹⁸ Rarely, JAK2 V617F mutations are found in patients with CNL.⁸

Although mutation analysis testing frequently uses bone marrow or peripheral blood cells, the Leumeta^® tests employed by Quest Diagnostics are performed using plasma samples (Table 2). For JAK2 mutation testing, plasma was shown to provide a higher mutation detection rate,¹⁹ to distinguish hemizygous/homozygous from heterozygous mutations,²⁰ and to detect mutations in RNA that might be missed with DNA cell-based testing.²¹ Furthermore, quantitation of JAK2 V617F allele burden in plasma permits true quantitation of leukemic burden, reporting in pg/μL plasma.

MPL Mutations

The myeloproliferative leukemia gene (MPL), found at chromosome 1p34, encodes the thrombopoietin receptor that works in concert with thrombopoietin for platelet production. Acquired MPL mutations (eg, W515L and W515K) are associated with severe anemia and have been detected in patients with ET or PMF but not in patients with PV (Table 4).^14,22 Typically, mutations in MPL are investigated after the JAK2 V617F mutation has been ruled out. An inherited MPL mutation (S505N; exon 10) has also been found in a Japanese pedigree with familial ET.²³

KIT Mutations

SM is associated with somatic activating point mutations within the KIT gene.⁷ The most common is mutation of the PTK domain at codon 816 (D816V). Detection of the D816V mutation satisfies a minor criterion for diagnosis of SM and predicts resistance to imatinib.²⁴

FIP1L1/PDGFRA Fusion Gene

The presence of the FIP1L1/PDGFRA somatic mutation confirms the diagnosis of FIP1L1/PDGFRA- associated CEL and predicts a favorable response to imatinib treatment.¹⁰ Favorable responses are also seen in a subset of patients with SM who are positive for FIP1L1/PDGFRA and eosinophilia (Table 4).

Immunophenotyping

Flow cytometric analysis for cell surface and cytoplasmic markers (immunophenotyping) is used to determine lineage for the differential diagnosis of leukemia and lymphoma and to distinguish benign from malignant processes. Such testing can be especially useful when transformation to AML is suspected. When a MPN progresses as reflected by increased aberrant cells, immunophenotyping helps guide therapeutic decisions. Decreases in abnormal marker-positive cells are associated with therapeutic success.

Chronic Myelogenous Leukemia Testing

Diagnosis of CML begins with testing for BCR/ABL1 in symptomatic patients or in those who have MPN-related abnormalities in their CBC (Figure 1). Fluorescence in situ hybridization (FISH) testing provides qualitative results for BCR/ABL1, whereas reverse transcription-PCR (RT-PCR) results are quantitative. Positive results are diagnostic of CML, while negative results eliminate CML. Other MPNs (BCR/ABL1-negative) should then be considered.

Figure 1. Differential diagnosis of the more common MPNs.

Approximately 95% of patients with CML are Ph+, whereas all are positive for BCR/ABL1 by FISH and RT-PCR. This seeming discrepancy can be explained by the presence of a masked Ph, which is not observed by conventional karyotyping but is detected by the molecular tests.²

Imatinib is the first-line therapy recommended for patients with chronic phase CML,²⁵ because it results in the lowest rate (~7%) of disease progression after 6 years of follow-up.²⁶ Bone marrow cytogenetics and quantitation of BCR/ABL1 transcripts are recommended prior to treatment (baseline assessment) and to assess therapeutic response (Table 5).²⁵ The goal of CML therapy is to achieve complete cytogenetic response (CCyR) (ie, the absence of Ph+ metaphase cells) within 18 months of start of therapy.²⁵ For molecular testing, RT-PCR is favored over FISH because BCR/ABL1  mRNA correlates with CCyR, and can be used to monitor the kinetics of leukemia disease burden. Most studies have found a good correlation between blood and marrow PCR values, thus avoiding bone marrow sampling.

As shown in Table 5, the testing performed and the frequency of testing depends on the clinical response to PTK inhibitor therapy. Monitoring begins with testing for the Ph chromosome and quantitating BCR/ABL1 transcripts. Decreasing levels indicate therapeutic success, while increasing levels may require more frequent monitoring and may be associated with an increased risk of therapeutic failure.

Imatinib therapy can result in acquired resistance, resulting from point mutation(s) in the kinase domain (KD) domain of BCR/ABL1 or insertion of a 35-nucleotide segment from intron 8 into the ABL1 KD.^27,28 Point mutations in particular may precede or accompany progression to a more aggressive disease.²⁹ ABL1 KD mutation testing is appropriate for patients presenting with advanced disease and for chronic-phase patients with inadequate initial response (Table 5). In addition, mutation testing is indicated when loss of response is apparent (eg, patient shows hematologic relapse, return of Ph+, or an increase in BCR/ABL1 transcripts).²⁵

Depending on the mutation, imatinib resistance can be overcome by increasing the dosage or by changing to second-generation PTK inhibitors, such as dasatinib and nilotinib. KD point mutations such as F311L, F359V, and L387M may be addressed by increasing the dosage, whereas M244V, G250E, and M351T may require switching to a second-generation drug.²⁷ However, the T315I KD mutation is associated with resistance to all 3 of these PTK inhibitors. Molecular modeling suggests that the BCR/ABL1^35INS mutation will cause resistance similar to the T315I mutation.²⁸ However, the degree of resistance may depend on the relative proportion of mutated vs wild-type BCR/ABL1 expression.²⁸

Both dasatinib and nilotinib have been successfully used in patients with CML who develop acquired resistance to imatinib.³⁰ However, resistance may develop with this second-line treatment, and, if so is usually associated with the emergence of new KD mutations.³⁰

Table 6 summarizes testing used in selecting and monitoring therapy and assessing prognosis for CML and other MPNs.

Essential Thrombocythemia

Although ET is diagnosed mainly by exclusion, a sustained platelet count of ≥450 x 10⁹/L, increased megakaryocyte proliferation in bone marrow studies, and presence of a JAK2 V617F mutation are consistent with the diagnosis (Table 1). Presence of the JAK2 V617F mutation can also serve to rule out CML, myelodysplastic syndrome (V617F is negative in up to 95% of patients), and non-hematologic cancer but not refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) (V617F positive in ~70%).^31-33 On the other hand, absence of a JAK2 mutation should be followed up with chromosome testing to rule out neoplasms such as CML and MDS associated with del(5q) (Figure 1).³

Normal C-reactive protein levels rule out reactive causes of thrombocytosis, which are more common than clonal ET, while PV is ruled out with normal hemoglobin and hematocrit. Fibrotic stage PMF is ruled out by the absence of reticulin and/or collagen fibrosis in bone marrow.

Thrombosis is the most common complication in patients with ET and can lead to death. JAK2 V617F mutation is associated with a ~2-fold increased risk of thrombosis.³⁴ In addition, the frequency of thrombosis and the risk of recurrence of a thrombotic event progressively increase with JAK2 V617F allele burden.^34,35

Disease progression to PMF or AML is unusual but can be detected by increased fibrosis in bone marrow or by increases in chromosomal abnormalities, respectively. Laboratory tests used to select and monitor therapy and to assess prognosis for ET are presented in Table 6.

Polycythemia Vera

Increased hemoglobin (>18.5 g/dL in men or >16.5 g/dL in women) is the hallmark of PV diagnosis.

The WHO recommendations for diagnosing PV include ruling out the more common inherited and secondary, acquired erythrocytosis.⁴ Acquired erythrocytosis can be due to chronic hypoxia, treatment with erythropoietin (EPO) or androgens, or EPO-secreting tumors. Once elevated hemoglobin levels are observed, a positive JAK2 mutation (either V617F or in exon 12) and a subnormal serum EPO level excludes secondary erythrocytosis and inherited polycythemia (see Figure 1). An elevated EPO level rules out PV, while a normal level is inconclusive. Negative JAK2 mutation makes the diagnosis of PV very unlikely but does not rule it out.

A high JAK2 V617F allele burden is associated with increased risk of cardiovascular events in patients with PV. Comparing the highest quartile allele burden with the lowest quartile revealed a 3.6-fold increase in risk of thrombosis.³⁶ The frequency of thrombosis progressively increased with amount of JAK2 V617F allele burden both at diagnosis and during follow-up.³⁶ After 5 years of follow-up post diagnosis, another study indicated that standard risk factors (eg, ≥60 years of age and previous thrombosis) lost their prognostic value and only JAK2 V617F allele burden predicted subsequent thrombosis.³⁴

Disease progression may manifest with post-polycythemic myelofibrosis or transformation to AML and/or MDS. Assessment of bone marrow fibrosis and chromosome analysis is used to identify these transformations. Additional laboratory tests to select and monitor therapy and to assess prognosis for PV are presented in Table 6.

Primary Myelofibrosis

The classical presentation of PMF is the appearance of a leukoerythroblastic blood smear with teardrop poikilocytosis, anemia, splenomegaly and possibly hepatomegaly due to EMH, and bone marrow fibrosis.⁵ This picture is characteristic of the fibrotic, advanced stage of the disease. Diagnosis is more complicated in the 20% to 30% of patients who are at the prefibrotic stage, which can resemble PV or ET. Observation of marked atypical forms of megakaryocytes, increased number of neutrophils, and decreased numbers of erythroid precursors in bone marrow confirm a diagnosis of prefibrotic PMF.⁵

Detection of the JAK2 V617F mutation supports the diagnosis of PMF but does not distinguish between PV, ET, or PMF. Absence of the mutation does not rule out the diagnosis. Detection of a clonal abnormality, which is relatively common in PMF (≈35%), or a MPL mutation also supports the diagnosis. A testing algorithm for the differential diagnosis of PMF is presented in Figure 1.

Table 6 summarizes tests used to monitor therapy and assess prognosis of PMF. A combination of age >65 years, hemoglobin levels <10 g/dL, WBC count >25 x 10⁹/L, ≥1% circulating blast cells, and the presence of constitutional symptoms predict shortened median survival.³⁷ Although del(20q) or del(13q) confer a survival advantage, typically the presence of an abnormal karyotype is associated with a poor prognosis.^37,38

Chronic Eosinophilic Leukemia

Hypereosinophilia (≥1.5 x 10⁹/L) may be due to reactive eosinophilia, idiopathic hypereosinophilic syndrome (HES), or CEL. The differential diagnosis of CEL and HES begins with the exclusion of all causes of reactive eosinophilia, including parasitic infection, infectious disease, allergic reaction, pulmonary diseases such as hypersensitivity pneumonitis, collagen vascular diseases, and underlying neoplastic disease.^6,9 Neoplastic diseases to be excluded include T-cell lymphomas, Hodgkin lymphoma, acute lymphoblastic leukemia/lymphoma, other MPNs, AML, and myelodysplastic syndromes. Once these diagnoses have been excluded, CEL is diagnosed if there is evidence of a clonal myeloid abnormality (eg, the presence of the FIP1L1/PDGFRA fusion gene) or increased number of blast cells (>2% in peripheral blood or >5% in the bone marrow). Figure 2 provides a testing algorithm for the differential diagnosis of hypereosinophilia.

Figure 2. Differential diagnosis of hypereosinophilia.

Table 6 summarizes tests used to select and monitor therapy and assess prognosis in patients with CEL or HES. Patients with idiopathic HES may be pre-leukemic; thus, monitoring is recommended.⁶

Systemic Mastocytosis

The major diagnostic criterion for SM is the presence of dense multifocal clusters or aggregates of mast cells (≥15 per aggregate) in a bone marrow biopsy specimen.⁷ Minor criteria include: 1) abnormal morphology in >25% of mast cells; 2) KIT mutation at codon 816; 3) mast cells coexpressing CD117 and CD2 and/or CD25; and 4) serum tryptase levels persistently >20 ng/mL in the absence of an associated hematologic clonal non-mast cell lineage disease. SM is diagnosed if at least the major criterion plus 1 minor criterion, or at least 3 minor criteria, are met (Table 1). A testing algorithm for SM diagnosis is presented in Figure 3. Diagnostic criteria for variants of SM are described in reference 7.

Figure 3. Differential diagnosis of systemic mastocytosis.

An elevated tryptase level is an important marker of SM but may also be seen in acute and chronic myeloid leukemias, other MPNs, myelodysplastic syndromes, and myelomastocytic leukemia.²⁴

In addition to being a criterion for diagnosis, the KIT D816V mutation confers resistance to imatinib by interfering with the binding of the drug to the catalytic site of the KIT PTK. Conversely, the presence of wild-type KIT, KIT F522C, or the FIP1L1/PDGFRA fusion gene is associated with sensitivity to imatinib.¹⁴

Table 6 summarizes tests used to select and monitor therapy and assess prognosis in patients with SM.

Chronic Neutrophilic Leukemia

CNL is diagnosed when the hematologic criteria are met (Table 1), hepatosplenomegaly is present, no evidence of a physiologic neutrophilia is found, and other MPNs and myelodysplastic disorders are ruled out.⁸ A normal C-reactive protein level rules out inflammation and infection that could produce neutrophilia; however, an elevated level does not necessarily rule out CNL. Negative results for Ph or BCR/ABL1 rule out CML, which also presents with neutrophilia. Other MPNs are ruled out by the absence of their usual hematologic characteristics (Table 1). Furthermore, myelodysplastic disorders are ruled out by the absence of granulocytic dysplasia and myelodysplastic changes in other myeloid lineages. A testing algorithm for CNL diagnosis is presented in Figure 4.

Figure 4. Differential diagnosis of chronic neutrophilic leukemia.

Disease progression may be reflected by transformation to AML, which can be detected by chromosome analysis. A summary of tests used to monitor therapy and assess prognosis of CNL is presented in Table 6.

MPN, Unclassifiable

In 10% to 15% of MPN cases, clinical and laboratory features characteristic of a myeloproliferative disease are present but fail to meet the diagnostic criteria of any one MPN.³⁹ Such patients are either in the early stages of the disease and characteristic features of a particular MPN will develop with time or are in the advanced stages of the disease with marked marrow fibrosis or blastic infiltration. In the former case, reevaluation at intervals of 4 to 6 months is recommended.³⁹